PROJECT SUMMARY Viruses, especially DNA viruses, elicit DNA damage responses (DDRs) in infected cells which are innate, critical barriers that can either impede or facilitate virus replication, and so must be successfully negotiated for infection to succeed. Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to enhance its infection in host cells. The Parvovirinae are small non-enveloped icosahedral viruses that are important pathogens in many animal species including humans. They are the only known viruses of vertebrates that contain single-stranded linear DNA genomes, and thus, present novel replicative DNA structures to cells during infection. They rely extensively on cellular processes for replication. This F32 application proposes a comprehensive and intensive training program whose scientific objective is to further investigate how MVM exploits the cellular DDR to prepare the nuclear environment for effective parvovirus takeover, focusing on the following aims: Aim 1: Identify where MVM establishes infection and characterize how its proximity to the host- genome changes temporally. Successful MVM replication leads to the formation of viral replication centers in the nuclear environment. It is not known where in the nucleus the virus establishes its replication bodies, and what part of the cellular genome it interacts with. We propose to identify the interactome of MVM replication bodies using chromosome conformation capture assays and how this association evolves throughout infection. Aim 2: Determine how MVM-NS1 aids in the establishment of viral replication centers. The MVM non-structural protein NS1, essential for viral replication, forms a core component of viral replication centers. Our preliminary data indicates that NS1 binds to cellular DNA at specific sites, perhaps assisting viral genomes as they establish replication centers at these cellular sites. Thus, we propose to identify the binding sites of NS1 throughout the cellular genome, and test the role of NS1 as a mediator of MVM-host interaction Aim 3: Determine how cellular DNA damage affects virus localization and replication. MVM infection elicits a DDR, leading to cell-cycle defects and the generation of cellular DNA breaks. Accumulation of DDR proteins at cellular sites can serve as hotspots for viral replication to occur. If this is true, engineered DNA breaks at selected sites would be predicted to recruit viral genomes. We propose to test the recruitment of MVM to engineered DNA breaks in a cell-line model, how genome recruitment changes temporally with DNA break induction, and characterize how DDR proteins accumulate over time at these sites. This work will add fundamental knowledge into how a small DNA virus negotiates the nuclear environment to initiate infection, and provide important information concerning how cells respond to the insult of incoming viral single-stranded DNA. Understanding these processes will elucidate how parvoviruses infect cells and ultimately how they cause disease, persist as gene therapy vehicles, or act as anti-cancer agents.